Optical sensor and optical method for characterizing a chemical or biological substance

ABSTRACT

The optical sensor contains an optical waveguide ( 1 ) with a substrate ( 104 ), waveguiding material ( 105 ), a cover medium ( 106 ) and a waveguide grating structure ( 101-103 ). By means of a light source ( 2 ), light can be emitted to the waveguide grating structure ( 101-103 ) from the substrate side and/or from the cover medium side ( 101-103 ). With means of detection ( 11 ), at least two differing light proportions ( 7-10 ) radiated from the waveguide ( 1 ) can be detected. For carrying out a measurement, the waveguide can be immovably fixed relative to the light source ( 2 ) and the means of detection ( 11 ). The waveguide grating structure ( 101-103 ) itself consists of one or several waveguide grating structure units ( 101-103 ), which if so required can be equipped with (bio-)chemo-sensitive layers. The sensor permits the generation of absolute measuring signals.

The invention concerns an optical sensor and an optical process for thecharacterization of a chemical and/or bio-chemical substance.

Waveguide grating structures with and without a chemo-sensitive layerare described in the literature (refer to, e.g., EP 0 226 604 B1, EP 0482 377 A2, PCT WO 95/03538, SPIE Vol. 1141, 192-200, PCT WO 97/09594,Advances in Biosensors 2 (1992), 261-289, U.S. Pat. No. 5,479,260, SPIEVol. 2836, 221-234).

In EP 0 226 604 B1 and EP 0 482 377 A2, it is demonstrated, how theeffective refractive index (resp., the coupling angle) of achemo-sensitive grating coupler can be measured as a sensor signal. Thesensor signal “effective refractive index” or “coupling angle” is avalue which manifests a strong dependence on temperature.

Front-side in-coupling of light into a waveguide (refer to SPIE Vol.1141, 192-200) is not practical, because a high positioning accuracy isrequired. In addition, the front side of the wave guide has to be ofgood optical quality. In PCT WO 95/03538 it is demonstrated how theabsolute out-coupling angle of a mode is measured. This value, however,without referencing manifests a high dependence on temperature. In PCTWO 97/09594, chirped waveguide gratings are presented, which, however,also manifest a dependence on temperature.

In Advances in Biosensors 2 (1992), 261-289 it is shown how thedisturbing “pore diffusion” can be referenced away with the three-layerwaveguide model. The refractive index of the waveguiding film manifestsdrift, while the layer thickness of the waveguiding film (=sensorsignal) remains stable. The layout is designed with movable mechanics,which does not permit rapid measurements. In addition, the sensor signalor the light emerging from the waveguide grating structure is recordedfrom the front side. Front side detection is unsuitable for atwo-dimensional layout of waveguide grating structure units.Furthermore, the effective refractive indexes N(TE) and N(TM) for thetwo polarizations TE and TM are not simultaneously recorded, because forthe recording of resonance coupling curves separated by angle amechanical angular scan is carried out.

In U.S. Pat. No. 5,479,260, a bi-diffractive or multi-diffractivegrating coupler is described, whereby the sensor signal is produced bythe interferometry of two out-coupling beams of the same or of differingpolarization (with the use of a polarizer). Interferometric measurementsare complicated, because the intensities of the two beams have to bematched to one another. In addition, temperature fluctuations due to theinterferometric signal generated by differing polarizations (using apolarizer) are only partially compensated.

In SPIE Vol. 2836, 221-234, a layout for a waveguide grating structurein connection with fluorescence- or luminescence measurements isdescribed. This layout, however, is not suitable for an (if necessarysimultaneous) (absolute) temperature-compensated measurement on thebasis of a direct detection. In addition, the waveguide gratingstructure is mounted on a revolving table.

In Applied Optics 20 (1981), 2280-2283, a temperature-independentoptical waveguide is reported about, whereby the substrate is made ofsilicon. Silicon is absorbent in the visual spectral range. In the caseof chemo-sensitive waveguide grating structures, however, thein-coupling takes place in preference from the substrate side. InApplied Optics 20 (1981), 2280-2283, in addition grating couplers whichare not temperature-independent are dealt with.

The invention presented here has the object of creating a(bio-)chemo-sensitive optical sensor and to indicate an optical processfor the characterization of a (bio-)chemical substance, which do nothave the above disadvantages. With the invention, in particular:

(1) sensor signals can be generated, which manifest a low dependence ontemperature and/or a low dependence on the diffusion of the specimenliquid into the micropores of a waveguiding film;

(2) both the measurement of (absolute) sensor signals with respect to adirect detection (absolute out-coupling angles α(TE) and α(TM) for theTE- or TM-wave, effective refractive indexes N(TE) and N(TM) for the TE-or TM-wave, layer thickness t_(F) of the waveguiding film etc.) as wellas the measurement of (absolute) sensor signals with respect to amarking detection (referenced fluorescence-, luminescence-,phosphorescence signals, etc.), are possible; and

(3) sensor signals remain stable with respect to a slight tilting and/ordisplacement of the waveguide grating structure, because (local andangular) differences of sensor signals or referenced sensor signals aremeasured. The object is achieved by the invention as it is defined inthe independent claims.

The optical sensor according to the invention contains at least oneoptical waveguide with a substrate, waveguiding material, a cover mediumand at least one waveguide grating structure, at least one light source,by means of which light can be emitted from the substrate side and/orfrom the cover medium side onto at least a part of the waveguide gratingstructure, and means for the detection of at least two differing lightproportions, whereby with at least one detection agent light emittedinto the substrate and/or cover medium can be detected, whereby for thecarrying out of a measurement the waveguide can be fixed immovably withrespect to the at least one light source and the means of detection.

In the case of the optical process according to the invention for thecharacterization of a chemical and/or bio-chemical substance in aspecimen by means of an optical waveguide containing at least onewaveguide grating structure, the specimen is brought into contact withthe waveguide in at least one contact zone, in the waveguide in theregion of the at least one contact zone at least one light wave isexcited through the waveguide grating structure, the at least one lightwave is brought into interaction with the specimen, light in at leasttwo differing proportions is detected, of which at least one proportionoriginates from the region of the contact zone, and at least oneabsolute measuring signal is generated by the evaluation of the detectedlight.

The waveguide grating structure consists of one or several waveguidegrating structure units, which are arranged one-dimensionally ortwo-dimensionally (e.g., in a matrix shape or circular shape).

A possible xy-displacement (or only an x-displacement) of the readinghead (the reading heads) from one waveguide grating structure to theother or a possible xy-displacement (or only x-displacement) of thewaveguide grating structure can quite well be applied.

A waveguide grating structure unit consists of at least two “sensingpads” (sensor platforms, sensor paths), which differ from one another inat least one of the following characteristics:

(a) The light waves guided in the “sensing pads” differ in theirpolarization (TE-wave or TM-wave), whereby the generated sensor signalis not produced by interferometric measurement.

(b) The light waves guided in the “sensing pads” differ in their modenumber.

(c) The two chemo-sensitive layers assigned to the “sensing pads”manifest a differing specificity (ligand 1 selectively binds (inside oron the surface) to the chemo-sensitive layer covering the “sensing pad1”; ligand 2 selectively binds (inside or on the surface) to thechemo-sensitive layer covering the “sensing pad 2”).

(d) The chemo-sensitive layer assigned to the first “sensing pad”manifests specificity for one ligand (with or without “non-specificbinding”), while the (chemo-sensitive) layer assigned to the second“sensing pad” manifests no specificity (with or without “non-specificbinding”) (example: Dextran layer, to which no identification molecule(e.g., an antibody) is bound).

(e) The light waves guided in the “sensing pads” differ in theirwavelength.

A “sensing pad”, in which guided light waves of differing polriztion(TE-wave or TM-wave) are excited, counts as two “sensing pads”(difference in the polarization!), providing the sensor signal generatedis not produced by interferometric measurement.

A “sensing pad”, in which guided light waves of differing mode numberare excited, counts as two “sensing pads” (difference in the modenumber!).

A “sensing pad”, in which guided light waves of differing wavelengthsare excited, counts as two “sensing pads” (difference in thewavelength!).

The first and second “sensing pad” can also be considered as signal- andreference path. The two “sensing pads” can (but do not have to) have thesame structure.

The (bio-)chemo-sensitive layer contacts the waveguiding film in acontact zone. This contact zone normally in the case of the directdetection contains at least one grating. (In the case of interferometricmeasurements with the same polarization, for a direct detection the(bio-)chemo-sensitive layer can also only be located between twogratings (also refer to EP 0 226 604)). (In the case of interferometricmeasurements with two differing polarizations (using a polarizer), the(bio-)chemo-sensitive layer can also be located on a multi-diffractive(bi-diffractive) grating (refer to U.S. Pat. No. 5,479,260)).

On principle, for example, the value S(signal path)−cS(reference path)can serve as a possible referenced sensor signal, whereby S(signal path)and S(reference path) are the sensor signals in the first “sensing pad”(signal path) or in the second “sensing pad” (reference path) and c is acalibration factor. In the case of the same polarization, sensibly c=1.In the case of different polarizations, with c the differingsensitivities of the two polarizations can be taken into account. In thecase of different wavelengths or mode numbers, with c the differingsensitivities of the wavelengths or of the mode numbers can be takeninto account. Advantageously, signal path and reference path are asclose together as possible.

The referenced sensor signal in the case of the same polarizationfurthermore has the advantage that disturbances δ, such as, e.g., thosecaused by temperature fluctuation, light wavelength fluctuation,undesired diffusion of molecules in the waveguides, resp. in thechemo-sensitive layer, unspecific bindings, fluctuations of theconcentration of the molecules not to be detected, etc., or combinationsof these, can be referenced away, i.e., the referenced sensor signal isindependent of δ, because S(signal path)+δ−(S(referencepath)+δ)=S(signal path)−S(reference path).

In preference, mono-mode waveguides are utilized, which only carry thefundamental TE-mode or only the fundamental TE-mode and the fundamentalTM-mode. The waveguiding film should preferably consist of ahigh-refractive material, which guarantees the generation of highsensitivities. The waveguiding film can be coated with a chemo-sensitivelayer (e.g., an anti-body layer (e.g., suitable for the detection of acorresponding antigen), a dextran layer with identification molecule(e.g., antibodies), receptors, DNA-sections, a silicon layer for thedetection of hydrocarbons, etc.). The waveguiding film itself, however,can also represent a chemo-sensitive layer. Rib waveguides can also beutilized.

A “sensing pad” comprises at least one grating, but can also comprise a(possibly more strongly modulated) in-coupling grating and at least oneout-coupling grating. The grating periods of in-coupling grating andout-coupling grating can be different (and are in most cases different).

In-coupling grating and out-coupling grating can be uni-diffractive ormulti-diffractive grating structures (bi-diffractive gratings, gratingswith changing grating period and/or with changing grating diffractionvector, etc.).

A preferred “sensing pad” arrangement consists of three gratings,whereby the middle grating represents the in-coupling grating and thetwo outer gratings two out-coupling gratings. With a strongly modulatedin-coupling grating, for example, one succeeds in exciting modes inforward and reverse direction with a sole (resp., with two) incident (ifso required slightly focussed) light beam(s). While a slightdisplacement of the incident light beam in the mode propagationdirection or a slight tilting of the waveguide grating structure withrespect to the incident light beam (in the plane of incidence) changethe intensity of the modes (running in forward and/or reverse direction)as a result of the changed coupling geometry, not, however, theout-coupling angle(s) or the difference of the out-coupling angles(=double absolute out-coupling angle), which represent possible sensorsignals.

FIG. 1 shows the above preferred “sensing pad” arrangement both for theTE-mode as well as for the TM-mode, whereby the two “sensing pads” arelocated adjacent to one another.

FIG. 2 illustrates further waveguide grating structure units.

FIG. 3 shows an optical sensor according to the invention.

A further preferred “sensing pad” arrangement also consists of threegratings, whereby the two outer gratings form (possibly more stronglymodulated) in-coupling gratings and the middle grating the out-couplinggrating.

In case of the detection of (bio-)molecular interactions with thearrangement according to FIG. 1 (or equivalent arrangements), thewaveguide structure unit is coated with a (bio-)chemo-sensitive layer,to which then in the experiment a specific binding partner binds, whichleads to a change of the out-coupling angles a(TE)=(α(TE+)−α(TE−))/2 andα(TM)=(α(TM+)−α(TM−))/2 (notation: e.g., α(TE+): out-coupling angle ofthe TE-mode running in +x-direction or to a change of the effectiverefractive indexes N(TE) and N(TM) and of the integrated optical valuesderivable from it—such as, e.g., of the layer thickness t_(F) of thewaveguiding film in the three-layer waveguide model (see further below).Different waveguide grating structure units can be coated with differentchemo-sensitive layers.

The out-coupling angle of the two out-coupling gratings (resp., of theout-coupling grating) of the preferred “sensing pad” arrangement enablean absolute determination of the out-coupling angle (resp., of theeffective refractive index), although the out-coupling of the two lightbeams does not take place at one and the same location.

In case of two adjacent “sensing pads” for the TE- and the TM-modeaccording to the preferred “sensing pad” arrangement (see FIG. 1), theout-coupling even takes place at four different locations.

The “sensing pad” for the TE-mode enables the determination of theeffective refractive index N(TE) of the TE-mode. The “sensing pad” forthe TM-mode enables the determination of the effective refractive indexN(TM) of the TM-mode. In case of simultaneous illumination of the two“sensing pads” (with, e.g., a single light field with 45° linearpolarized light or circular (or elliptical) polarized light), thedetermination of the out-coupling angle (of the effective refractiveindex) for the TE- and TM-mode can take place simultaneously.

FIG. 1 illustrates an advantageous embodiment of a waveguide gratingstructure unit. The “sensing pad” for the TE-mode consists of anin-coupling grating G_(i)(TE) and the two out-coupling gratingsG_(o+)(TE) und G_(o−)(TE) located on the left and right of thein-coupling grating. The sensing pad for the TM-mode consists of thein-coupling grating G_(i)(TM) and the two out-coupling gratingsG_(o+)(TM) and G_(o−)(TM) located on the left and right of thein-coupling grating and it is located adjacent to the “sensing pad” forthe TE-mode. In order to keep the influence of disturbances (e.g.,temperature fluctuations) low, the two “sensing pads” should be locatedas closely as possible adjacent to one another. The grating lines arealigned parallel to the y-axis.

It is advantageous if the out-coupling gratings G_(o+)(TE), G_(o−)(TE),G_(o−)(TM), G_(o−)(TM) are completely illuminated by the guided lightwaves, as a result of which it is ensured that in case of the respectiveout-coupling grating the sensor surface corresponds to the surface ofthe out-coupling grating. This is achieved by selecting (a) the width ofthe out-coupling grating in y-direction smaller than/equal to the widthof the in-coupling grating in y-direction and (b) the illumination spoton the in-coupling grating having a y-width, which is greater than/equalto the y-width of the out-coupling gratings, and the illumination spotincluding the complete y-width of an out-coupling grating.

An advantageous embodiment is the one, in the case of which the twoin-coupling gratings G_(i)(TE) and G_(i)(TM) are simultaneouslyilluminated with one (or two (wedge-shaped) light field(s) linearpolarized under 45°. With this, simultaneously the TE-mode and theTM-mode (in the forward and reverse direction) can be excited andsimultaneously all (in the case presented here: four) out-couplingangles can be measured. G_(i)(TE) and G_(i)(TM) in preference havediffering grating periods.

It is, however, also possible that a “sensing pad” as such carries outthe function of a “sensing pad” for the TE-mode and the function of a“sensing pad” for the TM-mode. The second “sensing pad” (locatedadjacent to it) is then either not present or else is utilized as acheck or reference. (This reference “sensing pad” can, e.g., be coatedwith a second chemo-sensitive layer or with a non-specific layer, i.e.,a layer which has no specificity (with or without “non-specificbinding”). If the in-coupling grating of a “sensing pad” is illuminatedwith TE-light as well as with TM-light under the correspondingin-coupling angles (e.g., with 45° linear polarized light), then in theone “sensing pad” both the TE-mode as well as the TM-mode are excited(if so required both in forward as well as in reverse direction).

It is also possible to excite the TE-mode only in forward direction andthe TM-mode only in reverse direction (or vice-versa) or the TE-mode andthe TM-mode only in forward direction (or vice-versa). In such a case,then the corresponding two out-coupling angles or their difference aremeasured.

Utilized as detectors are advantageously one- or two-dimensional(digital or analogue) position-sensitive detectors (PSD), photo-diodearrays, CCD line- or surface camera(s), etc. The out-coupled light beamsare advantageously focussed with a lens, whereby the focus does notindispensably have to be situated exactly on the detector surface. Theout-coupling gratings can also be focusing gratings. This has thebenefit that the lens-effect is already integrated in the out-couplinggrating.

It is also possible to select the direction of the grating lines of theout-coupling grating in such a manner, that the grating lines arevertical to the propagation direction of the mode. Advantageously, thetilting of the grating lines (resp., of the grating diffraction vectors)of the out-coupling gratings of two adjacent “sensing pads” has aninverse operational sign. With this, a better separation of theout-coupled light waves can be achieved.

The in-coupling grating can also be selected in such a manner, that thegrating period in x-direction or y-direction does not remain constant oris graduated. The requirements of the accuracy of the setting of theangle of incidence are relaxed as a result of this.

The in-coupling gratings shown in FIG. 1 in preference have a strongmodulation and are characterized by a short width in x-direction. As aresult, the requirements of the accuracy of the setting of the angle ofincidence are relaxed, because the light can now in-couple from agreater angle segment.

FIGS. 2a) and 2 b) illustrate two further embodiments of waveguidegrating structures, whereby here the dimension of the in-couplinggratings in y-direction is smaller than that of the out-couplinggratings. In FIG. 2b), a uniform out-coupling grating G_(O−) for the TE-and TM-wave running in (−x)-direction and a uniform out-coupling gratingG_(O+) for the TE- and TM-wave running in (+x)-direction are depicted.

FIG. 3 illustrates an embodiment of a sensor according to the invention.The sensor contains a waveguide 1 with a substrate 104, a waveguidingmaterial 105 and a cover medium 106 as well as with a waveguide gratingstructure according to FIG. 1. A light source 2 generates, for example,45° linearly polarized light; for this purpose, a linearly polarizedlaser with a polarization plane inclined to the drawing plane by 45° canbe utilized. Through a beam splitter 3 or a mirror 4, two impinginglight beams 5, 6 are produced. The light beams 5, 6 impinge on the twoin-coupling gratings 101 (of which in the side view of FIG. 3 only oneis visible) of the two “sensing pads” through the substrate 104 and inthe first “sensing pad” generate a TM-wave in forward- and reversedirection. The light waves are out-coupled through four out-couplinggratings 102, 103 (of which in the side view of FIG. 3 only two arevisible) from the waveguide grating structure and radiated into thesubstrate 104. After passing through the substrate 104 they spread aslight fields 7-10 and impinge on a two-dimensional CCD-detector 11. Ifso required, there can be a lens system (not illustrated) between thewaveguide 1 and the detector 11.

If in the two “sensing pads” one operates with differing polarizationand with a chemo-sensitive layer assigned to the waveguide gratingstructure, then, for example, the sensor signal S=t_(F) (=layerthickness of the waveguiding film in the three-layer waveguide model)can be generated (also see further below). If now a second waveguidegrating structure unit, which also permits the generation of the sensorsignal S=t_(F), is laid next to the first waveguide grating structureunit (now to be considered as a whole as signal path) and the secondwaveguide grating structure unit (now to be considered as a whole asreference path) coated with, for example, a chemo-sensitive layer ofdiffering specificity or with a non-specific chemo-sensitive layer withor without “non-specific binding” (e.g., a dextran layer, to which noidentification molecule is bound), then, for example, the value t_(F)(measured in the signal path)-t_(F) (measured in the reference path) canserve as sensor signal.

Although the effective refractive indexes N(TE) and N(TM) of the TE- orTM-mode are possibly not measured at the same point (the two “sensingpads” are, however, in the case of a chemo-sensitive sensor coated withthe same chemo-sensitive layer), with the waveguide grating structureunit the interesting sensor signals S and/or ΔS with

ΔS=Δα(TM)−Δα(TE)=Δ(α(TM)−α(TE))(ΔS=Δ(α(TE)−α(TM)))

(α=out-coupling angle)

ΔS=ΔN(TM)−ΔN(TE)(ΔS=ΔN(TE)−ΔN(TM)),

whereby the layer thickness of the waveguiding film in preference shouldbe selected in such a manner, that for the sensitivity of the refractiveindex of the cover medium ∂N(TE)/∂n_(C)=∂N(TM)/∂n_(C) is applicable,which at least eliminates the influence the refractive-index changes ofthe cover medium on the sensor signal have on the sensor signal (therefractive index nc of the cover medium depends on the temperature,i.e., n_(C)=n_(C)(T), whereby T is the temperature),

ΔS=Δt _(F)=const((∂N(TE)/∂T)⁻¹ ΔN(TE)−(∂N(TM)/∂T)⁻ ΔN(TM))

whereby const=((∂N(TE)/∂T)⁻¹(∂N(TE)/∂t_(F))−(∂N(TM)/∂T)⁻¹(∂N(TM)/∂t_(F)))⁻ and thechange of the layer thickness Δt_(F) is calculated from the system ofequations

 ΔN(TE)=(∂N(TE)/∂t _(F))Δt _(F)+(∂N(TE)/∂T)ΔT

ΔN(TM)=(∂N(TM)/∂t _(F))Δt _(F)+(∂N(TM)/∂T)ΔT  (1)

and ∂N/∂T is the temperature coefficient of the waveguide complete withcover (=specimen) for the corresponding mode (∂N/∂T is calculated takinginto account the temperature coefficient (dN/dT)_(GRATING) of thegrating experimentally (or according to theory) (also see furtherbelow)),

ΔS=Δt_(F), whereby the layer thickness t_(F) is calculated with thethree-layer waveguide model (with N(TE) and/or N(TM) as inputparameter),

ΔS=Δt_(A), whereby the change of the additional layer thickness Δt_(A)is calculated from the system of equations

ΔN(TE)=(∂N(TE)/∂t _(A))Δt _(A)+(∂N(TE)/∂T)ΔT

ΔN(TM)=(∂N(TM)/∂t _(A))Δt _(A)+(∂N(TM)/∂T)ΔT,  (2)

ΔS=Δt_(A), whereby the additional layer thickness t_(A) is calculatedwith the four-layer waveguide model (resp., five-layer waveguide model),

ΔS=ΔΓ, whereby the mass density by surface Γ (refer to Chem. Commun.1997, 1683-1684) is calculated with the four-layer waveguide model (oralso with the three-layer waveguide model with the utilization ofapproximations (refer to J. Opt. Soc. Am. B, Vol. 6, No.2, 209-220)),

etc., are measured. The above sensor signals are interesting, becausethey manifest a low dependence on temperature. On principle, however,during the measurement. it still has to be taken into account that alsothe grating period manifests a temperature-dependence as a result of thethermal expansion coefficient of the sensor chip. Applicable is ΔΛ=αΛΔT,whereby ΔΛ is the change of the grating period Λ, ΔT the change of thetemperature T and α the thermal expansion coefficient (typically 4.510⁻⁶ K⁻¹ for glass and 6.1 10⁻⁵ K⁻¹ for poly-carbonate). The(dN/dT)_(GRATING)=1(λ/Λ)α caused by the grating, whereby 1 is the orderof diffraction and λ the wavelength, contributes to the temperaturecoefficient (dN/dT)_(CHIP) (=experimental measured value) of thecomplete sensor chip as follows:(dN/dT)_(CHIP)=(dN/dT)_(WAVEGUIDE+SPECIMEN)+(dN/dT)_(GRATING), whereby(dN/dT)_(WAVEGUIDE+SPECIMEN) is the temperature coefficient of thewaveguide complete with cover (=specimen).

The sensor signals S and ΔS can be recorded in function of the time. Onecan, however, also only measure and compare an initial and a finalcondition on a waveguide grating structure unit, whereby, for example,in the interim other waveguide grating structure units can be evaluatedor the even the waveguide grating structure can be removed from themeasuring unit in the meantime, because absolute angles or differencesof angles (resp., distances (differences of distances) of light points)are measured and these values remain stable with respect to a tilting ordisplacement. The determination of a measured value can be carried outby means of an individual measurement, also, however, by means of astatistical evaluation (e.g., averaging) of several individualmeasurements.

If apart from temperature changes ΔT, other interferences are alsopresent, such as, e.g., diffusion of the specimen liquid into themicro-pores of a porous waveguiding film (which leads to a change in therefraction index Δn_(F) of the waveguiding film) or tensions (such as,e.g., compressive strain or tensile stress) or changes in stress arepresent or if in general only pore diffusion is present, then (in thefirst case under certain conditions (e.g., the course in function oftime of the pore diffusion with the temperature T as group of curves'parameter is known)) (approximately), then a (common) interference valueξ with N(t_(F),ξ)=N(t_(F), n_(F)(ξ), n_(S)(ξ), n_(C)(ξ)) or N(t_(A),ξ)=N(t_(F), t_(A), n_(A)(ξ), n_(F)(ξ), n_(S)(ξ), n_(C)(ξ)) can beintroduced. Equation (1) or (2) then take on the form

 ΔN(TE)=(∂N(TE)/∂S)ΔS+(∂N(TE)/∂ξ)Δξ

ΔN(TM)=(∂N(TM)/∂S)ΔS+(∂N(TM)/∂ξ)Δξ,  (3)

whereby ΔS is the sensor signal Δt_(F) (oder Δt_(A)) and, for example,in the case of the three-layer waveguide model∂N/∂ξ=(∂N/∂n_(F))(∂n_(F)/∂ξ)+(∂N/∂n_(S))(∂n_(S)/∂ξ)+(∂N/∂n_(C))(∂n_(C)/∂ξ)(+(∂N/∂t_(F))(∂t_(F)/∂ξ), providing t_(F) is=t_(F)(ξ)). Logically directly from ξ=Tformula (1) or formula (2) follows. ξ=n_(F), describes, e.g., thediffusion at a constant temperature.

Also in the case of the presence of several interferences, it ispossible to operate with the three-layer waveguide model and the modesequation solved (example: input: N(TE), N(TM), output: t_(F), n_(F)(=refractive index of the waveguiding film) or t_(F), n_(C) (=refractiveindex of the cover medium) or t_(F), n_(S) (=refractive index of thesubstrate)). In the three-layer waveguide model, waveguiding film,chemo-sensitive layer and specific binding partner are considered asbeing a layer with one refractive index. Although n_(F) and n_(C) (andif applicable n_(S)) are not precisely known and if applicable changeduring the experiment on the basis of an interference, the measuredvalue t_(F) is relatively independent of the interference, dependent onthe contrary, however, on the binding process. The interference isso-to-say packed into the second output value, the parameter “refractiveindex”.

Fundamentally, pore diffusion and temperature fluctuations areindependent manifestations and should be described by two interferencevalues ξ₁ and ξ₂ (generalization: k independent disturbances aredescribed by k independent disturbance values ξ₁, . . . ,ξ_(k)). FromN(t_(F), ξ₁, . . . ,ξ_(k)) for both polarizations, for a given modenumber and for a given wavelength follows

ΔN=(∂N/∂t _(F))Δt _(F)+(∂N/∂ξ ₁)Δξ₁+ . . . +(∂N/∂ξ _(k))Δξ_(k).  (4)

Because the disturbance values can influence the waveguide parameterst_(F), n_(F), n_(S), n_(C), the following is applicable:

∂N/∂ξ _(i)=(∂N/∂t _(F))(∂t _(F)/∂ξ_(i))+(∂N/∂n _(F))(∂n_(F)/∂ξ_(i))+(∂N/∂n _(S))(∂n _(S)/∂ξ_(i))+∂N/∂n _(C))(∂n_(C)/∂ξ_(i))  (5)

(1<=i<=k) (the analogue is also applicable for N(t_(A), ξ₁, . . .,ξ_(k)). For the pore diffusion (ξ₁=n_(F)) and the temperature change(ξ₂=T) from Eq. (4) results

ΔN=(∂N/∂t _(F))Δt _(F)+(∂N/∂n _(F))Δn _(F)+(∂N/∂T)ΔT  (6)

for both polarizations, a given mode number and a given wavelength.∂N/∂T can, e.g., be determined experimentally. If, e.g., bothpolarizations are measured, then (6) represents two equations with threeunknown values. If, however, measurements are carried out at severalwavelengths (taking into account the refractive index dispersions)and/or mode numbers, then several systems of equations respectivelyconsisting of three equations and three unknown values can be puttogether and solved. If the dispersion in Δn_(F)(λ) is taken intoaccount as an unknown value (λ=wavelength), then one obtains, e.g., withtwo wavelengths λ₁ and λ₂ and two polarizations four equations with fourunknown values Δt_(F), Δn_(F)(λ₁), Δn_(F)(λ₂), ΔT and from themdetermines the unknown values. The value Δt_(F) forms the sensor signal.

In analogy to further above, however, also the sensor signal t_(F) andthe disturbance values from the (three-layer- or four-layer-) modeequations for the two polarizations and for several wavelengths and modenumbers can be determined, this under the prerequisite, that the systemof equations can be numerically solved.

Fundamentally also the layer thicknesses are to a certain degreedependent on the temperature (also refer to Applied Optics 20 (1981),2280-2283), which fact, however, has been neglected in the formulas (1),(2) and (3) in the approximation. It is, however, also possible withrespect to at least one specimen select such a combination of (several)layers and substrate, that the temperature coefficient of the waveguideor of the grating coupler (resp., of the waveguide grating structure)with respect to at least one sensor signal S (=α(TE), α(TM),α(TM)−α(TE), N(TE), N(TM), N(TM)−N(TE), t_(F), t_(A) etc.) ispractically zero (the sensitivity, however, still remains high). Inthis, e.g., one of the layers involved can be an SiO₂ layer.

The absolute temperature coefficient with respect to a sensor signal Sis dS/dT, the corresponding relative temperature coefficient is(1/S)(dS/dT). with dN(TE)/dT=0 or dN(TM)/dT=0, also S=N(TE) or S=N(TM)become interesting sensor signals independent of the temperature.

If in a “sensing pad” only one grating is present, then, for example,with the reflection arrangement described in the European patentapplication 0 482 377 A2 (possibly with a sensor chip with tilting ofthe grating plane versus the lower substrate plane) measurements arepossible. The measurement with the reflection arrangement on weakly ormore strongly modulated (mono-diffractive or multi-diffractive)waveguide gratings (homogeneous gratings, superimposed gratings withdifferent grating periods and/or grating orientations, chirped gratings,etc.) can be carried out both for TE- as well as for TM-waves. The modeexcitation can take place from the left, the readout from the right orvice-versa. For example, in a “sensing pad” the excitation of theTE-wave can take place from the left and the excitation of the TM-wavefrom the right. In a second “sensing pad” (possibly with a differentgrating period and/or grating orientation) the excitation can be inreverse to the first “sensing pad”. The readout can take place in thereflected and transmitted light field in the zero-th or higherdiffraction order. With this, it is also possible to determine absolutecoupling angles with the reflection arrangement. The measured absolutecoupling angle, which in essence in case of the same grating period (andthe same diffraction order) corresponds to half of the angle differencebetween the two corresponding resonance minimums, does not change, evenif the sensor chip is slightly tilted. From the absolute coupling anglesfor the TE-wave or the TM-wave, the corresponding effective refractiveindexes and further integrated optical measured values can bedetermined, such as, e.g., the layer thickness d_(F) of the waveguidingfilm in the three-layer waveguide mode., etc.

It is of course also possible that in the first “sensing pad” theexcitation of the TE- and TM-wave takes place from the left and thereadout from the right (if so required with only one CCD) and in thesecond “sensing pad” (possibly with a differing grating period) inreverse.

It is also possible that the plane of incidence of the beam guidanceresponsible for the reference path (second “sensing pad”) is rotated ortilted or rotated and tilted versus the plane of incidence of the beamguidance responsible for the signal path (first “sensing pad”). Firstand second “sensing pads” can also coincide. The coupling angles for theTE-wave and the TM-wave can also be measured at different wavelengthsand/or mode numbers.

A further “sensing pad” arrangement consists of two (identical) chirpedgratings (gratings with graduated grating period), whereby one gratingserves as in-coupling grating and one grating as out-coupling grating.Chirped gratings are known from the literature (refer to, e.g., thepatent application WO 97/09594). The signal “sensing pad” and thereference “sensing-pad” can have the same or also an opposing chirpdirection (direction, in which the grating period changes (e.g., becomesbigger), it is vertical to the direction of the mode propagation). The“sensing pads” can be coated with the same (bio-)chemo-sensitive layer(see below) or else have differing ((bio-)chemo-sensitive) layers,whereby the ((bio-)chemo-sensitive) layer of the second “sensing pad”(reference “sensing pad”) is a different (bio-)chemo-sensitive layer ora non-specific ((bio-)chemo-sensitive) layer with or without“non-specific binding” (e.g., dextran without identification molecule)or else a purely protective layer.

The chirped grating, which is responsible for the in-coupling, isilluminated with a (not wedge-shaped or if so applicable (slightly)wedge-shaped) light strip, of which a certain proportion, in the case ofwhich the in-coupling conditions are fulfilled, is in-coupled to thewaveguide. It is also possible to simultaneously illuminate thein-coupling chirped gratings of the signal- ‘sensing pad’ and reference-‘sensing pad’ (if so required with a single, longer light strip) (if sorequired with 45° linear polarized or circular (or elliptically)polarized light (for the excitation of modes of both polarizations)). Inthe case of a excitation of modes of differing polarization under afixed angle of incidence, the corresponding grating periods of the two“sensing pads” are different.

In the case of the same polarization and opposite chirp direction ofsignal “sensing pad” and reference “sensing pad” (both “sensing pads”are coated with the same (bio-)chemo-sensitive layer), the twoout-coupled light spots on the basis of the (bio-)chemical bindingtravel (practically) vertical to the direction of propagation of themodes towards one another or away form one another (depending on thechirp direction or the chirp orientation). The position of the lightspots is measured with PSDs or with a one- (or two-) dimensional CCD.Through the change of the distance of the two light spots, the change ofthe effective refractive index of the corresponding polarization can becalculated. The distance of the two light spots is an absolute value,because the distance of the two light spots is independent ofdisplacements or of small tilting. (If the signal and reference pathshave a differing polarization, then the measuring signal ΔN(TM)−ΔN(TE)can be determined.)

In the case of the same polarizations, same chirp directions of the two“sensing pads”, but differing (bio-)chemo-sensitive layer (e.g., on thesignal path a specific layer, on the reference path a non-specificlayer), the distance of the two light spots forms a referenced(absolute) sensor signal.

The arrangement described in the paragraph before the last one can onceagain be duplicated for the other polarization (with an adaptation ofthe grating period) and can once again as a whole be considered asreference path for the complete layout described in the paragraph beforethe last one (to be interpreted now as signal path). Here the change ofthe effective refractive index of the other polarization can bemeasured. (If, however the polarization(s) remains (remain) the same andinstead another chemo-sensitive layer (with or without non-specificbinding) or a non-specific layer with or without non-specific binding(e.g., dextran layer without identification molecule) is utilized, thenthe referenced measured values (measured value (firstarrangement)-measured value (second arrangement)) of the measured valuesΔN (TE) or ΔN(TM) or ΔN(TM)−ΔN(TE) can be determined).

It is also possible, that only the in-coupling grating is present as achirped grating, the out-coupling grating, however, e.g., ismono-diffractive (or multi-diffractive). The two light spots describedthree paragraphs above do not anymore travel vertically to the modepropagation direction on the basis of the (bio-)chemical binding,because the out-coupling grating also deflects in the plane of incidence(possibly better referred to as the outlet plane) (i.e., theout-coupling angle changes).

If on the other hand for signal “sensing pad” and reference “sensingpad” one operates with three gratings each (one in-coupling grating andtwo out-coupling gratings or two in-coupling gratings and oneout-coupling grating), whereby modes in forward and reverse directionsare excited, if the in-coupling grating is a chirped grating (with thesame or with opposite chirp orientation between the two “sensing pads”)and if the in-coupling gratings are, e.g., mono-diffractive (or multidiffractive), then displacements or tilting of the x- and y-axis(orientation of the axis as in FIG. 1) can be identified and eliminatedon the basis of the absolute measurement. For the chemo-sensitivelayer(s), the same remarks are applicable as in the case of the “sensingpad”, which consists of two chirped gratings. If, for example, only the(absolute) out-coupling angle (and/or measured values which can bederived from it) are considered as sensor signals, then the chirpedin-coupling grating can also be illuminated with a light strip impingingin a wedge-shape. Here too, the above arrangement (signal and referencepath) can be duplicated and considered as a new reference path (with orwithout differing chemo-sensitive layer or with a non-specific layer) tothe above arrangement (now as a whole as signal path).

The specimen liquid(s) is (are) brought into contact with the waveguideor with the chemo-sensitive substance(s) through a “well” or a matrix of“wells”, a through-flow cell or a matrix of through-flow cells, acapillary vessel or a matrix of capillary vessels.

Two-dimensional arrangements lead, e.g., to a “microplate” with 96, 384,1536 “wells”, etc. (But also other two-dimensional formats (e.g., disks)are possible). But also the manufacturing of individual strips(one-dimensional arrangement) is possible. The strips can, e.g., also beinserted into the frame of a microplate. The “wells” can be affixed tothe sensor-chip plate, which contains the waveguide grating structureunits as separate specimen cell (resp., specimen cell plate). It ishowever, also possible to provide the substrate itself with indentationsin such a manner, that these indentations already assume the function ofthe “wells” or of the through-flow vessels or of the capillary vessels.In the two latter cases, the sensor-chip plate has to be covered with acovering plate equipped with bores. In the first case, the sensor-chipplate can be covered with a covering plate (without bores), in order to,e.g., prevent evaporation. The bores serve for the supply or carryingaway of the specimen or for the ventilation. In preference, one workswith plastic material and injection-moulding techniques or hot-embossingtechniques. But also sol-gel techniques, UV-hardening techniques fororganic/inorganic composites, glass embossing techniques (hot-embossingor (injection-)moulding), etching techniques, material removal by laser,etc., present an alternative.

The grating structures can be manufactured using embossing techniques(hot-embossing, cold-embossing, UV-hardening) or injection-mouldingtechniques out of plastic material, sol-gel, glass, UV-hardening organicor inorganic materials or organic/inorganic composites, ormoceres ornanomeres, with removal by laser in conjunction with interferometry,holography and/or phase mask techniques, photo-lithography inconjunction with wet or dry etching, using photo polymerization (referto, e.g., P. Coudray et al., Crit. Rev. Opt. Sci. Tech. (SPIE) CR68(1997), 286-303) or using casting techniques (e.g., in sol-gel), etc.Plastic injection-moulding techniques, such as are utilized in themanufacturing of compact discs (e.g., out of poly-carbonate), areparticularly suitable. The grating structure can be in (resp., on) thesubstrate or in (resp., on) a layer or can be present in a combinationof these. The grating structures can be surface relief gratings (resp.,interface relief gratings) or refractive-index gratings (resp., volumegratings) or combinations of these. The waveguiding film can be asol-gel layer (SiO₂—TiO₂, TiO₂, (non-porous) high-refractionlead-silicate glass, etc)., an organic/inorganic composite layer, apolymer layer, a PVD-, a CVD- a PE-CVD-, a PI-CVD layer, aphoto-polymerizable, high-refraction layer (e.g., photo-polymerizableTiO₂), etc. or combinations of these. It is also possible that a layer(with in preference low refractive index, made, e.g., of SiO₂ sol-gel ,SiO₂—TiO₂ sol-gel (with low TiO₂ content), lead-silicate glass, sol-gel(with low lead content), sol-gel similar to float-glass, etc.) containsthe grating structure and a second layer (e.g., made of SiO₂—TiO₂, TiO₂,Ta₂O₅, HfO₂, ZrO₂, Nb₂O₅, Si₃N₄, lead-silicate glass, etc.) forms thewaveguiding film. Due to the fact that the softening point oflead-silicate glass is significantly below the softening point of glass,the lead-silicate glass can be made to melt without damaging the glassin doing so. The melting process leads to a practically pore-freelead-silicate glass. If the substrate and the layer(s) have similarthermal expansion coefficients, then the formation of micro-fissures canbe prevented (micro-fissures increase the dampening values of themodes). PVD- and CVD-processes make possible the manufacture of verycompact waveguiding films.

The substrate can be made of a plastic material (e.g., poly-carbonate,PMMA, polystyrol, etc.), sol-gel or of glass (float-glass, specimenslides, soda-lime glass, boro-silicate glass, alkali-free glass, quartz,etc,). However, also the grating material can be utilized as substratematerial (e.g., ormocere, UV-hardening material).

The specimen cell can consist of indentations (holes, bores), also,however, of through-flow cells. These through-flow cells can also beconfigured in such a manner, that the specimen liquid can be suppliedusing the pin of a pipette robot. It is also possible, that a secondspecimen cell consisting of through-flow vessels is inserted into afirst specimen cell with indentations (holes).

In the substrate or anywhere on the sensor-chip plate, positioning marks(holes, indentations, pins, elevations, etc.) can be inserted oraffixed. These positioning marks guarantee that the impinging light beamhits the in-coupling grating(s). The identification of the positioningmarks is effected through the measuring unit.

For the separation of the light wave carrying the sensor signal fromother light waves (e.g., from the light wave reflected from the bottomof the substrate), it is also advantageous if the waveguiding film isnot plane-parallel to the bottom of the substrate. Thisnon-plane-parallelism can be present, among others, in the form ofwedges, prisms, cylinder prisms, spherical lenses, cylinder lenses, etc.

With the chemo-sensitive waveguide structure, not only a directdetection, but also marking detections can be carried out. Thutilization of a “refractive index label” (e.g., of a little plasticball (e.g., a small latex ball), biochemical and biological fragments,etc.), can already be considered as a marking detection. With it, forexample, sandwich assays or competition assays can be implemented.

As light source a monochromatic light source, such as, e.g., a (pulsed)laser, a (pulsed) laser-diode, a (pulsed) LED with or without filter inthe (infra)red or blue-green or ulta-violet spectral range, isadvantageously used. But also thermal light sources with a filter can beutilized. Red-blue-green or ultra-violet wavelengths have the advantagesthat (a) the sensitivity for the direct detection increases and (b) withthe same light source apart from “direct sensing” also fluorescence-,phosphorescence- or luminescence tests can be conducted, whereby theexcitation of the fluorescence, phosphorescence or luminescence takesplace through the evanescent wave (in the form of a TE-wave or TM-waveor TE-and TM-wave). The fluorescence-, phosphorescence- or luminescencelight can be observed as a plane wave or also as a guided wave. Thefluorescent light caused by the TE exciting wave (resp., its intensity)can be referenced or compared with the fluorescent light (resp., itsintensity) caused by the TM exciting wave, whereby advantageously theintensity of the exciting wave is jointly taken into account andpossibly the detectors are made sensitive to polarization by theutilization of polarizers. The guided fluorescence-, phosphorescence- orluminescence light wave can be out-coupled through a grating andconveyed to a detector. Sandwich-assays, competition-assays, etc., canbe carried out, whereby at least one participating binding partner isfluorescence(phosphorescence-, luminescence-) marked. The(bio-)chemo-sensitive layer can be present on the grating, can also,however, be present solely between the gratings or outside the grating.

Fluorescence, phosphorescence or luminescence measurements manifest alow dependence on temperature. For fluorescence, phosphorescence orluminescence measurements, purely inorganic waveguide grating structuresare particularly suitable (the grating is manufactured in glass orsol-gel (e.g., SiO₂) or in the inorganic waveguiding film, waveguidingfilm made of inorganic material (e.g., Si₃N₄ or oxide layers such as,e.g., TiO₂ or Ta₂O₅ or lead-silicate layers, etc.). Inorganic materialsmanifest, e.g., a low fluorescence of their own. If one works with aplastic material substrate, then it is recommended to apply an inorganiclow-refraction intermediate layer (e.g., SiO₂) to the plastic substrate.The layer thickness of this intermediate layer has to be selected asgreat enough, so that the evanescent wave running in it practically doesnot anymore “see” the plastic substrate. As a result of this, at leastthe proprietary fluorescence generated by the guided light wave isstrongly reduced.

If both the intensity of the (possible out-coupled) excitation wave aswell as that of the (possibly out-coupled) emission wave are measured,then various interference factors (such as, e.g., those, which arecaused by intensity fluctuations of the exciting wave) can be eliminatedby referencing. The referenced sensor signal is then, for example, theintensity of the fluorescence (phosphorescence, luminescence) divided bythe intensity of the exciting light wave. The intensity of the excitingwave can be measured prior to the penetration of the exciting wave intothe chemo-sensitive layer or after the exit of the exciting wave fromthe chemo-sensitive layer or at both points. With referenced markingdetections, absolute kinetic measurements as well as absolute end pointmeasurements can be carried out. In the case of absolute (end point)measurements, the (bio-)chemical interaction on the waveguide gratingstructure can also take place outside the measuring instrument.

It is also to be noted that with a (digital or analogue) PSD (positionsensitive detector) (one-dimensional or two-dimensional), aphoto-diode-array (PDA) (one-dimensional or two-dimensional) or aCCD-array (one-dimensional or two-dimensional), not only (positions of)light field distributions but also intensities can be measured.

It is possible (but it is not absolutely necessary) to in part with thesame detectors carry out a direct detection as well as a markingdetection (on a fluorescence, phosphorescence or luminescence basis).Evanescence field excitation can also be combined with measuringtechniques resolved over time (e.g., time-resolved fluorescence orluminescence). In the case of time-resolved measuring techniques, themarking is excited with a pulsed light wave (primarily in the visible orultra-violet wavelength range). The fading times for the fluorescence(luminescence, phosphorescence) of free and bound marking molecules(with or without energy transfer between two different fluorescencemolecules (refer to, e.g., the Förster theory in CIS Bio International,November 1995, No. 3)) are different. The depth of penetration of theguided excitation wave into the specimen determines the “observationvolume”.

It is interesting that with the waveguide grating structures presentedhere absolute measurements can be carried out both free of marking aswell as fluorescence (phosphorescence, luminescence) marked (if sorequired also simultaneously). In both cases, for the in-coupling oflight one can make do without movable mechanics. It goes without sayingthat also (continuous) kinetic measurements or real-time measurementscan be carried out on a non-absolute basis.

With a “sensing pad” (signal path) (with or without chemo-sensitivelayer), it is also possible to carry out light absorption measurements,inasmuch as the intensities of the light beams out-coupled (throughgrating, prism, taper or front side) (if so required with the samedetectors) can be measured, possibly also at different wavelengths. Thechange in light absorption can come about directly or indirectly (e.g.,through enzymes) through the (bio-)chemical interaction of the specimenwith the chemo-sensitive layer or through the specimen itself or throughthe reactions taking place in the specimen (with or without anadditional reaction partner). The chemo-sensitive layer can be presenton the grating, between the gratings or outside the grating.Fluctuations in light intensity of the light source can be eliminated byreferencing (e.g., with a beam-splitter and a reference detector orthrough a not in-coupled diffraction order and a reference detector).Referencing can also be carried out by the method in which the second“sensing pad” (reference path) is covered with a protective layer andtherefore cannot interact with the specimen. The referenced sensorsignal is then the intensity of the signal path detector divided by theintensity of the reference path detectors.

The protective layer, however, can also be a (porous) (chemo-sensitive)layer, which has no specificity (with or without “non-specific binding”)or specificity for another ligand.

Referencing can also (but does not have to) take place through one halfof a “sensing pad” (e.g., signal path: mode in forward direction,reference path: mode in reverse direction).

What is claimed is:
 1. An optical sensor for the characterization ordetection or characterization and detection of a chemical or biochemicalor chemical and bio-chemical substance comprising at least one opticalwaveguide with a substrate, a waveguiding material, a cover medium andat least one waveguide grating structure, at least two sensing padscomprising at least one waveguide grating for acting as in-couplinggrating, at least one of the sensing pads acting as sensor pad and beingat least partially coated by a sensor chemosensitive orbio-chemosensitve layer, and at least one of the sensing pads acting asreference pad and being coated at least partially by a referencechemosensitive or bio-chemosensitive layer, light source means for thesimultaneous illumination of at least the in-coupling gratings of thesensor pad and of the reference pad from the substrate side or from thecover medium side or from the substrate side and from the cover mediumside, detection means for simultaneous detection of positions orintensities or positions and intensities of at least two lightdistribution proportions, which, on the detection means, are notsuperimposed on one another and which are emitted or coupled out oremitted and coupled out from the waveguide grating structure into thesubstrate or into the cover medium or into the substrate and into thecover medium, means for the generation of a referenced sensor signalthrough the evaluation of the detected light distribution, or of thedetected positions or of intensities of the at least two lightdistribution proportions or of a combination of these, and means for theimmovable fixation of the waveguide grating structure relative to thelight source means and the means of detection for the purpose ofcarrying out a measurement.
 2. The optical sensor according to claim 1,wherein the sensor pad and the reference pad each comprise at least onein-coupling grating and at least one out-coupling grating.
 3. Theoptical sensor according to claim 1, wherein a lens system is arrangedbetween the waveguide and the detection means.
 4. The optical sensoraccording to claim 1, wherein the waveguide grating structure is anon-chirped grating structure.
 5. The optical sensor according to claim1, wherein at least one sensing pad of the waveguide grating structureunit contains at least one chirped grating.
 6. The optical sensoraccording to claim 1, comprising one detector for the detection of theat least two light proportions, said detector comprising means forresolving the positions and/or the intensities of the at least two lightproportions.
 7. The optical sensor according to claim 1, wherein thedetector is a 1-dimensional or a 2-dimensional photodiode array or CCDcamera or a 1-dimensional or 2-dimensional analogue position sensitivedetector or a 1-dimensional or 2-dimensional digital position sensitivedetector.
 8. The optical sensor according to claim 1 wherein a waveguidegrating structure comprises means for producing at least four emittedand/or out-coupled light fields corresponding to the forward and therearward direction of the transverse electric mode and the transversemagnetic mode.
 9. The optical sensor according to claim 1 comprisingmeans for conducting at least two of a direct sensing test and of afluorescence test and of a luminescence test and of a phosphorescencetest using only one light source.
 10. The optical sensor according toclaim 1, wherein the two sensing pads are adjacent to one another. 11.The optical sensor according to claim 1 wherein the two sensing pads arelocated in another or one above the other.
 12. The optical sensoraccording to claim 1 wherein the grating or gratings of the sensor padhave the same grating period or grating periods as the grating orgratings of the reference pad.
 13. The optical sensor according to claim1 wherein sensor pad and reference pad differ from each other in thegrating period of at least one grating.
 14. The optical sensor accordingto claim 1 wherein at least one grating is situated in the volume and/oron a bordering surface of a material contained in the waveguide.
 15. Theoptical sensor according to claim 2, wherein one sensing pad of thewaveguide grating structure unit contains two out-coupling gratings andan in-coupling grating situated between the out-coupling gratings, ortwo in-coupling gratings and an out-coupling grating situated betweenthe in-coupling gratings.
 16. The optical sensor according to claim 1,wherein a “well” or a matrix of “wells” is affixed onto the waveguidegrating structure or is inserted into the waveguide grating structure.17. The optical sensor according to claim 1, wherein a flow-through cellor a matrix of flow through cells or a capillary vessel or a matrix ofcapillary vessels are affixed onto the waveguide grating structure or isinserted into the waveguide grating structure.
 18. The optical sensoraccording to claim 1, wherein at least the reference chemosensitive orbio-chemosensitive layer shows essentially no non specific binding. 19.The optical sensor according to claim 1, wherein the sensor pad and thereference pad are arranged at least partially in a distance from eachother.
 20. The optical sensor according to claim 1, wherein the lightsource means are arranged and designed in a manner that an in-couplinggrating of the sensor pad and an in-coupling grating of a reference padcan be illuminated simultaneously.
 21. An optical process for thecharacterization or for the detection or for the characterization andfor the detection of a chemical or bio-chemical substance or a chemicaland bio-chemical substance in a specimen by means of a waveguide gratingstructure containing at least one waveguide grating structure unit,wherein: the specimen is brought into contact with the waveguidestructure in at least one contact zone comprising a sensorchemosensitive or bio-chemosensitive layer and a referencechemosensitive or bio-chemosensitive layer in the waveguide structure inthe region of the at least one contact zone, at least two light wavesare simultaneously excited through the waveguide grating structure unit,the light waves differing in at least one of their polarization, theirmode number, their wavelength and of their position on the waveguidegrating structure, the at least one light wave is brought intointeraction with the specimen, light is detected in at least twodiffering proportions, which are not superimposed on the detection meansand of which at least one proportion originates from the at least onecontact zone, and at least one referenced measured signal is generatedby the evaluation of the detected light.
 22. The process according toclaim 21, wherein the measured signal is generated on the basis of adirect detection.
 23. The process according to claim 22, wherein themeasured signal is a layer thickness or a change in layer thicknessaccording to the solution of the mode equation for at least onepolarization, at least one wavelength and at least one mode number. 24.The process according to claim 22, wherein the measured signal is alayer thickness or a change in layer thickness according to the solutionof a linear system of equations for at least one polarization, at leastone wavelength and at least one mode number.
 25. The process accordingto claim 21, wherein the measured signal is generated on the basis of amarking detection.
 26. The process according to the claim 21, whereinboth a measured signal belonging to the direct detection as well as ameasured signal belonging to the marking detection are generated. 27.The process according to claim 21, wherein the measured signal isgenerated resolved in function of time.
 28. The process according toclaim 21, wherein a waveguide structure or a waveguide grating structureis selected in such a manner, that the temperature coefficient of thewaveguide structure or of the waveguide grating structure is practicallyzero with respect to at least one specimen and with respect to at leastone measured signal.